The road to generating transplantable organs: from blastocyst complementation to interspecies chimeras

ABSTRACT Growing human organs in animals sounds like something from the realm of science fiction, but it may one day become a reality through a technique known as interspecies blastocyst complementation. This technique, which was originally developed to study gene function in development, involves injecting donor pluripotent stem cells into an organogenesis-disabled host embryo, allowing the donor cells to compensate for missing organs or tissues. Although interspecies blastocyst complementation has been achieved between closely related species, such as mice and rats, the situation becomes much more difficult for species that are far apart on the evolutionary tree. This is presumably because of layers of xenogeneic barriers that are a result of divergent evolution. In this Review, we discuss the current status of blastocyst complementation approaches and, in light of recent progress, elaborate on the keys to success for interspecies blastocyst complementation and organ generation.

Efficiency comparison of B6(Cg)-Tyrc−2j /J and C57BL/6NTac embryos as hosts for the generation of knockout mice

Careful selection of the host embryo is critical to the efficient production of knockout (KO) mice when injecting mouse embryonic stem (mES) cells into blastocysts. B6(Cg)-Tyrc−2j/J (B6 albino) and C57BL/6NTac (B6NTac) strains of mice are widely used to produce host blastocysts for such procedures. Here, we tested these two strains to identify an appropriate match for modified agouti C57BL/6N (JM8A3.N1) mES cells. When comparing blastocyst yield, super-ovulated B6NTac mice produced more injectable blastocysts per female than B6 albino mice (8.2 vs. 5.4). There was no significant difference in birth rate when injected embryos were transferred to the same pseudopregnant recipient strain. However, the live birth rate was significantly higher for B6NTac blastocysts than B6 albino blastocysts (62.7% vs. 50.2%). In addition, the proportion of pups exhibiting high-level and complete chimerism, as identified by coat color, was also significantly higher in the B6NTac strain. There was no obvious difference in the efficiency of germline transmission (GLT) when compared between B6NTac and B6 albino host embryos (61.5% vs. 63.3% for mES clones; 64.5% vs. 67.9% for genes, respectively), thus suggesting that an equivalent GLT rate could be obtained with only a few blastocyst injections for B6NTac embryos. In conclusion, our data indicate that B6NTac blastocysts are a better choice for the microinjection of JM8A3.N1 mES cells than B6 albino blastocysts.

An improved foolproof device for eggs’ sterilization of Corcyra cephalonica (Stainton)

Mass rearing of numerous biological control agents depends on large amounts of eggs of factitious hosts like rice meal moth, Corcyra cephalonica (Stainton) (Lepidoptera : Pyralidae). Corcyra eggs sterilization is required before they are used for parasitisation by Trichogrammatids. To meet the requirement of eggs sterilization of this insect, a new innovative device was invented, fabricated and studied. It consisted of an enclosed structure having semi-circular shaped drawer. An ultra violet (UV) tube of 30 W is fixed on the centre of its ceiling to maintain a uniform distance of 35 cm from the eggs to be sterilized. An exposure of 10 minutes to eggs of C. cephalonica was required to get optimum sterilization. As whole body of the device was an enclosed structure, hence, there was no exposure of UV rays to the laboratory workers. The different sized-models (150,100, 50 of “egg cards” required for Trichogrammatids) can be fabricated to meet the diverse requirements. Irradiated eggs were found having no any adverse effects on the abilities of Trichogrammatids parasitisation and its emergence. It can also used for UV sterilization of laboratory materials like glassware, plastic ware, clothes, cotton etc. The period of exposure to the UV light was set using timer. This innovative UV chamber had numerous advantages i.e., the UV rays used had damaged the host embryo. The damaged embryo could not develop into next stage, at the same time damaged embryo found suitable for parasitization of Trichogrammatids. The whole body of the device was made-up of an enclosed struc-ture; hence, there was no exposure of UV rays to the laboratory workers. The different sized-models (150,100, 50 of “egg cards” required for Trichogrammatids) can be fabricated to meet the diverse requirements of clients. As the UV chamber was made up of metal and ply boards, hence, it was durable. The longitudinal flanges provided in the drawer of the device prevented the host egg bearing cards to glide one over other from curved surface. The device was found safe and effective for sterilization of Corcyra eggs and easy to operate.

Stem cell potency and the ability to contribute to chimeric organisms

Mouse embryonic chimeras are a well-established tool for studying cell lineage commitment and pluripotency. Experimental chimeras were successfully produced by combining two or more preimplantation embryos or by introducing into host embryo cultured pluripotent embryonic stem cells (ESCs). Chimera production using genetically modified ESCs became the method of choice for the generation of knockout or knockin mice. Although the derivation of ESCs or ESC-like cells has been reported for other species, only mouse and rat pluripotent stem cells have been shown to contribute to germline-competent chimeras, which is the defining feature of ESCs. Herein, we describe different approaches employed for the generation of embryonic chimeras, define chimera-competent cell types, and describe cases of spontaneous chimerism in humans. We also review the current state of derivation of pluripotent stem cells in several species and discuss outcomes of various chimera studies when such cells are used.

90 GERM-LINE TRANSMISSION OF DONOR MITOCHONDRIAL DNA IN NUCLEAR TRANSFER-DERIVED COWS

In embryos derived by nuclear transfer (NT), fusion, or injection of donor cells with recipient oocytes caused mitochondrial heteroplasmy. Previous studies have reported varying patterns of mitochondrial DNA (mtDNA) transmission in cloned calves. Distribution of donor mtDNA found in offspring of NT-derived founders may also vary from donor–host embryo heteroplasmy to host embryo homoplasmy. Here we examined the transmission of mtDNA from NT cows to their progeny. NT cows were originally produced by fusion of enucleated oocytes with Jersey (J) or Holstein (H1) oviduct epithelial cells, or Holstein (H2) or Japanese Black (B) cumulus cells, as previously reported (Goto et al. 1999 Anim. Sci. J. 70, 243–245; Yonai et al. 2005 J. Dairy Sci. 88, 4097–4110; Akagi et al. 2003 Mol. Reprod. Dev. 66, 264–272). Transmission of donor cell mtDNA was analyzed by PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis of the mitochondrial D-loop region. Eleven NT founder cows were analyzed, 4 (2 = J-NT, and 2 = H1-NT) of them were heteroplasmic whereas 7 (1 = J-NT, 1 = H1-NT, 2 = H2-NT, and 3 = B-NT) were homoplasmic for the host embryo mitochondria. The proportions of donor mtDNA detected in one J-NT cow was 7.7%, and those of other cow lineages were <2%. Heteroplasmic NT cows delivered a total of 9 progeny. Four of the 9 progeny exhibited heteroplasmy with high percentages of donor cell mtDNA populations (52%, 37%, 17%, and 43%). The other 5 progeny were obtained from heteroplasmic NT cows, and all samples of the 10 progeny obtained from the homoplasmic NT cows did not harbor detectable donor cell mtDNA. A genetic bottleneck in the female germ-line will generally favor the transmission of a single mitochondrial population, leading to a return to homoplasmy. Thus, some of progeny maintained heteroplasmy with a higher ratio than that of their NT mothers, which may also reflect a segregation distortion caused by the proposed mitochondrial bottleneck. These results demonstrated that donor mtDNA in NT cows could be transmitted to progeny with varying efficiencies, in a lineage-specific fashion.

171 NORMAL REPROGRAMMING OF IMPRINTING IN PARTHENOGENETIC FEMALE GERM CELLS

Mammalian parthenotes cannot develop normally to term. Mouse parthenogenetic embryos die by Day 10 of gestation. On the other hand, viable parthenogenetic chimeras were produced by normal host embryos, although parthenogenetic cells were observed in a limited number of tissues and organs and, even in these instances, their contribution was substantially reduced. This can be explained by the aberrant expressions of imprinted genes in parthenogenetic cells. In female mice, erasure of imprints occurs around the time that primordial germ cells enter the gonad, and establishment of imprints occurs in the postnatal growth phase of oogenesis. In this study, we investigated whether aberrant imprints in parthenogenetic embryonic stem (PgES) cells can be erased through the germline. Diploid parthenogenetic embryos were produced by activation of (CBA × C57BL/6-EGFP) F1 mouse superovulated unfertilized oocytes by exposure to Sr2+ and cytochalasin B. Ten parthenogenetic blastocysts were plated and three PgES cell lines were isolated. Chimeras were made by injecting 10–15 PgES cells into ICR(CD-1) mouse blastocysts. Chimeras and chimeric tissues were detected by fluorescent microscopy. In all, 173 chimeric blastocysts were transferred to 9 recipient females, and 101 live pups containing 9 female and 21 male chimeras were born. No significant growth retardation was apparent in PgES chimeras, irrespective of their degree of chimerism. In 5 male chimeras killed at 1 day postpartum (dpp), PgES cells showed a restricted tissue contribution. The contribution to lung, liver, and intestine was considerably lower than in the other tissues such as brain, heart, spleen, and kidney. PgES derived or host embryo derived non-growing oocytes were isolated from dissociated ovaries of female chimeras at 1 dpp under fluorescent microscopy. Methylation imprints in non-growing oocytes were analyzed for maternally methylated imprinted genes Peg1, Snrpn, and Igf2r by the combined bisulfite restriction analysis (COBRA). In normal oocytes, imprints are expected to be erased and these genes are unmethylated at this stage. We observed that these genes were unmethylated in both PgES derived and host embryo derived non-growing oocytes. These results suggest that aberrant imprints in PgES cells can also be erased normally through the germline.